Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies

Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85.

Subgroup D avian sarcoma and leukosis viruses can penetrate a variety of mammalian cells in addition to cells from their natural host, chickens. Sequences derived from the gp85-coding domain within the env gene of a mammal-tropic subgroup D virus (Schmidt-Ruppin D strain of Rous sarcoma virus [SR-D RSV]) and a non-mammal-tropic subgroup B virus (Rous-associated virus type 2) were recombined to map genetic determinants that allow penetration of mammalian cells. The following conclusions were based on host range analysis of the recombinant viruses. (i) The determinants of gp85 that result in the mammal tropism phenotype of SR-D RSV are encoded within the 160 codons that lie 3' of codon 121 from the corresponding amino terminus of the gp85 protein. (ii) Small linear domains of the SR-D RSV gp85-coding domain placed in the subgroup B background did not yield viruses with titers equal to that of the subgroup D virus in a human cell line. (iii) Recombinant viruses that contained subgroup D sequences within the hr1 variable domain of gp85 showed modest-to-significant increases in infectivity on human cells relative to chicken cells. A recombinant virus that contained three fortuitous amino acid substitutions in the gp85-coding domain was found to penetrate the human cell line and give a titer similar to that of the subgroup D virus. In addition, we found that the subgroup D virus, the mutant virus, and recombinant viruses with an increased mammal tropism phenotype were unstable at 42 degrees C. These results suggest that the mammal tropism of the SR-D strain is not related to altered receptor specificity but rather to an unstable and fusogenic viral glycoprotein. A temperature sensitivity phenotype for infectivity of mammalian cells was also observed for another mammal-tropic avian retrovirus, the Bratislava 77 strain of RSV, a subgroup C virus, but was not seen for any other avian retrovirus tested, strengthening the correlation between mammal tropism and temperature sensitivity

Rearrangements in unintegrated retroviral DNA are complex and are the result of multiple genetic determinants.

We used a replication-competent retrovirus shuttle vector based on a DNA clone of the Schmidt-Ruppin A strain of Rous sarcoma virus to characterize rearrangements in circular viral DNA. In this system, circular molecules of viral DNA present after acute infection of cultured cells were cloned as plasmids directly into bacteria. The use of a replication-competent shuttle vector permitted convenient isolation of a large number of viral DNA clones; in this study, over 1,000 clones were analyzed. The circular DNA molecules could be placed into a limited number of categories. Approximately one-third of the rescued molecules had deletions in which one boundary was very near the edge of a long terminal repeat (LTR) unit. Subtle differences in the patterns of deletions in circular DNAs with one versus two copies of the LTR sequence were observed, and differences between deletions emanating from the right and left boundaries of the LTR were seen. A virus with a missense mutation in the region of the pol gene responsible for integration and exhibiting a temperature sensitivity phenotype for replication had a marked decrease in the number of rescued molecules with LTR-associated deletions when infection was performed at the nonpermissive temperature. This result suggests that determinants in the pol gene, possibly in the integration protein, play a role in the generation of LTR-associated deletions. Sequences in a second region of the genome, probably within the viral gag gene, were also found to affect the types of circular viral DNA molecules present after infection. Sequences in this region from different strains of avian sarcoma-leukosis viruses influenced the fraction of circular molecules with LTR-associated deletions, as well as the relative proportion of circular molecules with either one or two copies of the LTR. Thus, the profile of rearrangements in unintegrated viral DNA is complex and dependent upon the nature of sequences in the gag and pol regions